PROJECT SUMMARY This application describes broadly based biophysical characterization of two important aspects of bacterial cell physiology. First, the dynamics of the entire E. coli proteome will be quantified as a function of growth rate and nutrient limitation, including measurement of protein turnover. The set of bacterial proteins are carefully controlled to optimize the fitness and growth of bacteria under a wide variety of conditions. The amount of protein devoted to various biosynthetic pathway varies in a defined way that can be simply modeled as an economic allocation problem. Second, the process of ribosome assembly will be investigated using electron microscopy, mass spectrometry, single molecule fluorescence, and light microscopy. Electron microscopy of intermediates that accumulate under perturbed conditions reveals a distribution of structures that can be ordered into a putative assembly pathway. Single molecule fluorescence allows the monitoring of individual steps in the complex assembly process. Finally, we will extend the in vitro studies toward studying the ultrastructure of the locus of ribosome assembly in cells, using a combination of light microscopy and cryo-electron tomography. The overall goal is to develop a complete mechanistic framework for assembly of the largest cellular machine, that is responsible for all protein synthesis, and that requires almost a third of the energy budget for a rapidly dividing cell.